External ID: 2153-01_Inhibitor_Dose_DryPowder_Activity_Set9

Protocol: Protocol:

The susceptibility of the Dd2 Plasmodium falciparum strain against novel small molecules will be determined using a SYBR green-based fluorescence assay in 384-well plates.

1. Parasites are synchronized at ring stage using the sorbitol method before performing the assay.

2. 50 ul of parasite culture at 1.0% parasitemia and 1% hematocrit (O-positive human erythrocytes) is transferred into each well of 384-well plate

3. 10 nl of serially diluted compounds are transferred to plates.

4. Assay plates are transferred to a Thermo three-gas incubator for a further 72 hours incubation at 37 degrees Celsius in a low oxygen gas environment (1% O2, 4.1% CO2, balance N2).

5. 10 ul of SYBR lysis buffer (Saponin 0.16%, Tris-HCl (pH 7.5) 20 mM, EDTA (disodium salt) 5 mM, Triton X-100 6% v/v, SYBR Green I (Invitrogen) 1000X) is added to the each well of an assay plates.

6. Plates are incubated 20-36 hrs at room temperature before reading the SYBR green fluorescence intensity (EX 480 nm, EM 530 nM) using an Envison.

7. EC50 values are determined by regression analysis performed in the Collaborative Drug Discovery database system.

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC; n=28) and positive control wells (PC; n=18) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.

External ID: 2130-01_HTS

Protocol: Compounds (160 nL of stock solutions) were arrayed into clear, square well 384-well plates (Aurora, EB 3030-00330) using an Echo 555 acoustic dispenser, with neutral (DMSO) and positive controls (Levofloxacin, final concentration 16 mug/mL). For single-point primary screening final compound concentration was 16 ug/mL. Prior to screening, 25 muL of fresh growth medium was added to each well. C. difficile cultures were prepared by dispensing 1 mL from a thawed cryovial into 200 mL Bacto BHI with 2 % Oxyrase. The thawed cultures were grown for 18 hours in an anaerobic chamber at 37 degrees C; overnight cultures were diluted 1:100 in fresh medium and 25 muL of the prepared inoculum was added to each well. The inoculated plates were briefly centrifuged to remove any bubbles and incubated for 24 hours. After incubation, plates were removed from the incubator and equilibrated at room temperature for 30 minutes. OD600 was determined in an Analyst GT (Molecular Devices).

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: The plate pattern correction algorithm 'Assay Pattern (multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer and multiplied by -1 to conform with Pubchem Activity Score conventions.
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 80.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:

Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

External ID: 7059-01_Inhibitor_SinglePoint_HTS_Activity

Protocol: 1. AH1F cells are plated at 10000cells per well and allowed to adhere for 1hour 30ul volume.
2. Compounds are pinned.
3. Using combi 5ul of 7X (168nM) solution of Alexa 488-Fibronectin is added to the wells to final concentration of 24nM in DMEM Media with 2% serum is added to all the wells. Positive control wells did not receive labeled FN.
4. Cells are incubated overnight
5. Plates are washed 3 times with HBSS
6. 25ul of PBS is added
7. Plates are read using envision excitation 485nM/ emission 525

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: The plate pattern correction algorithm 'Assay Pattern (additive)' in Genedata (v10.0.2) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at -30%.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 50.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: 7157 Fluorescence anisotropy probe displacement-based high-throughput screen for exo-site inhibitors of Insulin-Degrading Enzyme (IDE)

Protocol: The human IDE(42-1019) gene was cloned into expression vector pTrcHis-A (Invitrogen) and transformed by heat-shock into chemically-competent expression strain Rosetta 2 (DE3) pLysS E. coli cells (EMD Millipore), selected on carbenicillin/chloramphenicol LB agar, and induced with IPGT overnight at 37 degrees C growing in 2xYT media. The protein was purified through Ni-NTA affinity column using the N-terminal 6x poly-histidine tag as previously reported (Maianti et al, Nature 2014, 511(7507): 94-8). Purified protein was stored at 4 degrees C in the standard FP buffer 50 mM Tris pH 8.0, with 1 M NaCl, with additional 1 mM TCEP and used within one week.

Fluorescence anisotropy high-throughput screening assay:
Fluorescein-labeled macrocycle FL-6b probe (30 nM final) and human N-His6-IDE(42-1019) (0.5 microM final concentration) were mixed in buffer containing 50 mM Tris buffer pH 8.0 and 1 M NaCl, chilled on ice and allowed to warm to room temperature. For a run of 50 x 384 well plates we used 1,000 mL of freshly prepared enzyme-probe solution in FP buffer, and used within 1 hour. The reliability of the Z'-factor was checked by pinning 6bK positive controls and DMSO negative controls each day of screening. The first pilot screen using the "DOS informer set" compound collection was transferred by pinning (100 nL/well) into 384-well plates (Corning FB Black #3575) using a CyBio Vario liquid handling system equipped with a pin-transfer workstation into wells preloaded with the enzyme-probe mixture (50 microL/well) transferred using a Multidrop Combi nL Reagent Dispenser (Thermo Scientific). For the second screen using azetidine-core libraries the compounds were transferred into empty 384-well plates by sonication (100 microL/well) using the Labcyte ECHO and the enzyme-probe mixture was added using the Combi nL Dispenser (50 microL/well, Corning FB Black #3575 plates). The final compound concentrations were 20 microM in both screens, and IDE inhibitor 6bK was used as a positive control at 1 microM final concentration. After 30 min equilibration at room temperature, fluorescence anisotropy was recorded using an EnVision spectrophotometer (excitation 492 nm, emission 523 nm). Exclusion of compounds using fluorescence measurements was not necessary for the screens performed up to date because the DOS compound libraries were designed to avoid fluorophores.

Comment: Activity Outcome
Active (2): ZScore <= 3
Inactive (1): ZScore >3

External ID: 7056-01_Inhibitor_SinglePoint_HTS_Activity

Protocol: Bac rRNA Automation Protocol
Day 1:
1. Grow each E. coli strain (one wild type (GFP) and six humanized mutants (RFP) including the control G2058) individually. Pick each strain into a separate 50 mL conical tube with 10 mL of LB medium supplemented with 100 ug/mL amplicillin and 50 ug/mL spectinomycin.
2. Incubate strains overnight at 37 degrees with shaking at 190 RPM.
Day 2:
1. In the morning, check OD600 of the cultures.
2. Dilute the cultures to OD 600 = 0.05 in LB medium supplanted with 100 ug/mL ampicillin.
3. Return strains to 37 degrees with shaking at 190 RPM for about 3. 5 hours. Grow strains to reach OD 600 about 0.5 (exponential growth phase).
4. Mix strains (final OD600= 0.02) in the proportion 50% wild-type, 8.33% each of the six mutants in LB medium supplemented with 100 ug/mL ampicillin and 1 mM IPTG (which is used to induce expression of fluorescent proteins).
5. Dispense 30 uL/well of bacteria mixture to 384 well Assay Ready Plates (ARPs) containing positive controls (Erythromycin 15 mg/mL, 50 nL in column 1 and Choramphenicol 15 mg/mL, 50 nL in column 23).
6. Place breathable film on plate using Plate Loc.
7. Incubate plates in Steristore at 37 degrees, 0% CO2 for 24 hours, 95% humidity.
Day 3:
1. Remove assay plate from Steristore.
2. Remove breathable film with X-Peel.
3. Read GFP signal using EnVision plate reader (mirror: FITC #403, Ex: FITC 485 #102, Em: FITC 535 #206).
4. Read RFP signal using EnVision plate reader (mirror: BODIPY TMR #405, Ex: photometric 550 #312, Em: Cy3 595 #229).
5. Read OD600 signal using EnVision plate reader (Ex: Photometric 600 #319).
6. Store plate in Steristore incubator.
Equipment Required
Combi
Plate Loc
Steristore (37 C, 95% humiodity, 0% CO2,)
X-Peel
2 EnVisions

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control
wells (PC) were included on every plate. One positive control,
Chloramphenicol, was used to determine the baseline fluorescence and OD
as it was expected to non-selectively kill all 6 strains, GFP wild-type
and RFP mutant. The other positive control, Erythromycin, was expected
to selectively spare one RFP control strain (G2058) and was therefore
used as a guideline for calculating the performance of a selective GFP
inhibitor.

NORMALIZATION:
Each of three layers was independently normalized using the 'Neutral
Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to
a normalized activity value of 0.
The a raw signal of 0 was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: The plate pattern correction algorithm 'Assay
Pattern (multiplicative)' in Genedata (v7.0.3) was applied to the
normalized plate data for each layer.

AGGREGATE COMPOUND LAYER SCORING: In addition to the reported normalized
percent activities above, each replicate compound measurement was
converted to a z-score relative to the corresponding DMSO-control
distribution, as described for ChemBank (Seiler et al., 2008). ChemBank
composite Z scores were computed to combine replicates for each channel
(RFP, GFP, and OD). We sought compounds that affected the GFP-expressing
wild-type strain while not affecting at least one of the mutant RFP
strains and not affecting overall viability as measured by OD; i.e.,
they score with negative composite Z in the GFP channel with little or
no effect in the RFP or OD channels. To combine the results of the three
channels quantitatively, we defined a trigonometric scaling function to
express our preferences that (1) GFP composite Z scores be negative, and
(2) RFP and OD composite Z score amplitudes exceed GFP composite Z score
amplitudes; such compounds should fall left of the DMSO-control
distribution, but above the main diagonal in a plot of GFP composite Z
versus either OD or RFP composite Z. Using the two ratios of ChemBank
composite Z scores (GFP/RFP and GFP/OD), we defined q1 = arctan(GFP/RFP)
and q2 = arctan(GFP/OD) and expressed our scale factor as F =
cos(-q1)cos(2q1)cos(-q2)cos(2q2), with the four factors respectively
expressing our preferences. As a three-assay composite score, we take
-(ZGFP) x F, which clearly discriminates the compounds of interest when
plotted against RFP or OD.

PUBCHEM_ACTIVITY_SCORE: The above three-assay composite score was
normalized on a scale of 1-100, with 1 being those with the least
desirable observed score representing compounds increasing in GFP signal
and decreasing in RFP or OD, and 100 being the most ideal observed score
representing a loss in GFP with no change in RFP or OD.

PUBCHEM_ACTIVITY_OUTCOME: The above three-assay composite scores were
fit to a standard distribution and a corresponding Holm-Bonferroni
corrected p value significance was calculated. Compounds with a FDR
corrected p value less than 0.05, normalized OD percent score > -30,
negative GFP composite Z score, and the expression Abs(Cos(-q)Cos(2q)) >
(sqrt2)/2 for both q1 and q2 were assigned a score of 2 (active).
Compounds failing any of these criteria were assigned a score of 1
(inactive.)

External ID: 7144-01_Activator_SinglePoint_HTS_Activity

Protocol: 7144- Mouse alpha TC1 cell qPCR

Media
DMEM low glucose 1 g/L
Hyclone FBS 10%
Pen/Strep 1%

Day 1 (Cell Plating)
Mouse alpha TC1 cells are added to each well at 6,000/well in a Corning CellBIND Black, 384-well, clear bottom, tissue culture plate in 50 uL of plating media using a standard Combi liquid dispenser (Thermo). The plated cells are then incubated overnight in a tissue culture incubator (Thermo) at 37 degrees C and 5% CO2

Day 2 (Compound Pinning)
The compounds are pinned into assay plates using a 384 well pin tool (100 nL) (Cybi Well). Pins are washed with methanol and DMSO between each pinning. Cell plates are returned to the incubator.

Day 5 (Media Exchange, Compound Pinning)
To make sure cell health and compound state are optimal, cell media is refreshed and compounds added again to assay plates. The plating media is aspirated from the cells and 50 uL of media is dispensed using a Biotek 406 plate washer. The compounds are re-pinned using 384 well pin tool (100 nL) (Cybi Well). Pins are washed with methanol and DMSO between each pinning. Cell plates are returned to the incubator.

Day 8 (cDNA Synthesis)
Cell lysis (using Cells-to-Ct kits, Ambion cat.no. 4391851C)
The medium is aspirated and the cells are washed twice (100 uL with PBS) using the ELX405 Plate washer (Biotek). The assay plates are de-lidded, flipped upside down and centrifuged at 1,000 rpm for 2 minutes to remove excess liquid. 10 uL of Lysis solution containing DNase I (from Cell to CT Lysis kit, Ambion) is added to each well using the MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific). Each assay plate is then shaken for 2 minutes and incubated for an additional 8 minutes at room temperature. At the end of the incubation, 1 uL of stop solution (from Cells to CT Lysys kit, Ambion) is added with the Multidrop Combi-nL (Thermo Scientific) and the assay plates are centrifuged face up (1,000 rpm, 2minutes) . The assay plates are incubated for 2 minutes at room temperature and processed for reverse transcription.

Reverse transcription (RT) (using Cell to CT RT kit, Ambion cat. no. #4402958)
10 uL RT Master Mix/12.5 uL total reaction volume)

2X RT Buffer, 6.25 uL
20X RT Enzyme Mix, 0.625 uL
Nuclease-Free Water, 3.125 uL

10 uL of RT master mix is dispensed into each well of a RT assay plate (Axygen, PCR-384 RGD C). 2.5 uL of the lysed cells are transferred into RT assay plate using Vario transfer unit (CyBi Well). The RT assay plates are incubated at 37 degrees C for 1h and the reverse transcriptase is inactivated by incubating the plates for 1 minute at 95 degrees C. cDNA is stored at -80 degrees C until ready for qPCR analysis.

Day 9+ (qPCR)
Prepare qPCR master mix (using Light Cycler Probes Master, Roche cat. no.4887301001)

4 uL PCR Master Mix/5uL total volume of reaction

2X Roche Master Mix (Probes Master), 2.5 uL
60X FAM Taqman probe/primers set (Mouse PAX4, Applied Biosystems, 4331182 (Mm01159036_m1)), 0.0416 uL
20X VIC Taqman probe/primers set (mouse GAPDH, Applied Biosystems 4352339E), 0.125 uL
PCR H2O, 1.33 uL

4 uL/well of PCR master mix is dispensed in PCR plate (Roche Light Cycler 480 MultiWell Plate 384, Cat# 04 729 749 001) using the Multidrop Combi-nl (Thermo Scientific).

1 uL/well of RT DNA is transferred to the 4 uL/well PCR plate using CyBi Well.

Prepare positive control (plasmid containing the mouse coding sequence of Pax4, "Pax4IRESGFP" 4700ng/mL stock) by diluting to 12 pg/uL in water. Dispense 1 uL in each well of column 1 and column 23 of qPCR plate using Multidrop Combi-nl (Thermo Scientific).

Plates are sealed with clear seal and are centrifuged (face up) 2 minutes at 1000 rpm.

PCR is performed using ThermoCycler (Roche Light Cycler 480 II) with Macro Probe Protocol with this setup:
95 degrees for 10 minutes
95 degrees for 10 seconds, 60 degrees for 30 seconds (repeat 55 times)
40 degrees for 30 seconds

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the maximum of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 15.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 50.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: Bursicon-induced LGR2 mediated cAMP production in LGR-2/CRE6x-Luciferase co-transfected HEK293 cells Inhibition - 7011-01_Antagonist_SinglePoint_HTS_Activity

Protocol: Protocol

Day 0:
Cell Culture
HEK 293 cells are plated in DMEM culture media (containing 10% Fetal Bovine Serum (FBS) and 1X penn/strep/glutamine) at a density of 5x106 cells/T175 flask and grown for 3 days at 37 degrees C in 5% CO2 incubator.

Day 3:
Transient Transfection
Cells are transiently transfected in DMEM transfection media (containing 1X penn/strep/glutamine without serum) using polyethyleneimine (PEI).
1)Mix 2.9ug/flask of cDNA encoding the wild-type Bursicon Receptor with 14.6ng/flask of cDNA encoding a CRE-luciferase reporter gene with a PEST/HCL sequence in 2mL of transfection media
2)Add 61uL/flask of PEI of 1mg/mL in 1.5mL of transfection media
3)Mix the 2mL cDNA solution with 1.5mL of PEI solution and incubate at RT for 20 mins
4)Aspirated the culture media from the cells in T175 flask
5)Add 25 mL of transfection media and 3.5mL of transfection mixture to T175 falsk.
6)Mix the media with transfection mixture well and transfect for 2 days at 37 degrees C in 5% CO2 incubator

Day 5:
Cell Plating
1)Typsinize the Cells with 5mL of 0.05% Trypsin and incubate at 37oC for 5 min.
2)Add 5mL of DMEM assay media (containing DMEM without phenol red, 10% NuSerum and 1x Pen/Strep/Glutamine) to inactive trypsin
3)Collect cells by centrifugation at 1000rpm for 5 min
4)Suspend the cells in DMEM assay media to a cell density of 4x105 cells/mL
5)Plate the cells to 1536-well Aurora Mako plate with 2000 cells/well/5uL using ViaFill
6)Incubate the cells at 37 degrees C in 5% CO2 incubator on GS system for overnight

Day 6:
Compound Treatment, Agonist Stimulation and Detection
Assay Setup
1)Thaw aliquoted Bursicon conditioned media and diluted with DMEM assay media at a 1/20 dilution. Filter the solution through a 0.22uM filter
2)Add DMEM condition media, the diluted Bursicon conditioned media and SteadyGlo to the bottles and setup BioRAPTR

Run automation protocol on GS system
1)Take the assay plate with transfected cells from the incubator
2)Transfer 5nL/well of 10mM compound to assay plate
3)Incubate 30 mins in incubator
4)Add 1uL/well of DMEM assay media to PosCon wells and diluted Bursicon solution to the other wells using BioRAPTR (Beckman) based on the plate map
5)Incubate the assay plate for 4 h in incubator
6)Take the plate out from the incubator and equilibrate the assay plate at RT for 10 mins
7)Add 1uL/well of SteadyGlo to assay plate using BioRAPTR, incubate at RT for 10 mins
8)Read the plate on ViewLux (PerkinElmer) for Luminescence.

Culture Media
DMEM high glucose, no glutamine (Invitrogen, 11960)
Pen/Strep/Glutamine (Invitrogen 10378-016)
Fetal Bovine Serum (Atlanta Biological, S10350)

Transfection Media
DMEM high glucose, no glutamine (Invitrogen, 11960)
Pen/Strep/Glutamine (Invitrogen 10378-016)

Assay Media
DMEM no phenol red (Invitrogen, 21063-029)
Pen/Strep/Glutamine (Invitrogen 10378-016)
NuSerum (BD biosciences, 24883)

0.05% Trypsin-EDTA (Invitrogen, 25300-062)
Aurora 1536-well Mako plate (Aurora/Brooks, 00028778)

Comment:

External ID: 2153-05_Inhibitor_SinglePoint_HTS_Activity

Protocol: The susceptibility of the Dd2 Plasmodium falciparum strain against novel small molecules will be determined using a SYBR green-based fluorescence assay in 384-well plates.

1. Parasites are synchronized at ring stage using the sorbitol method before performing the assay.

2. 50 ul of parasite culture at 1.0% parasitemia and 1% hematocrit (O-positive human erythrocytes) is transferred into each well of 384-well plate

3. 10 nl of serially diluted compounds are transferred to plates.

4. Assay plates are transferred to a Thermo three-gas incubator for a further 72 hours incubation at 37 degrees Celsius in a low oxygen gas environment (1% O2, 4.1% CO2, balance N2).

5. 10 ul of SYBR lysis buffer (Saponin 0.16%, Tris-HCl (pH 7.5) 20 mM, EDTA (disodium salt) 5 mM, Triton X-100 6% v/v, SYBR Green I (Invitrogen) 1000X) is added to the each well of an assay plates.

6. Plates are incubated 20-36 hrs at room temperature before reading the SYBR green fluorescence intensity (EX 480 nm, EM 530 nM) using an Envison.

7. EC50 values are determined by regression analysis performed in the Collaborative Drug Discovery database system.

Comment:

External ID: 7124-01_Inhibitor_SinglePoint_HTS_Activity

Protocol:
Protocol:
1) Plate 10 uL BL2-NFkB-luc cells at 12.5K cells/well, using a Multidrop Combi reagent dispenser (1,250 cells/uL).
2) Add 25 nL compound or positive control, by pin transfer; IKK inhibitor VII will be used at 50 uM.
3) Incubate at 37 masculineC for 1 hour (allowing compounds to enter cells for action).
4) Add 5 uL 192 ng/mL tCD40L per well for a final concentration of 64 ng/ml using a Thermo Multidrop Combi.
5) Incubate at 37 masculineC for 4 hrs, then leave plate at RT for 30 minutes.
6) Add 15 uL SteadyGlo luciferase substrate (Promega) per well using a Thermo Multidrop Combi.
7) Incubate at RT for 5min, then read Luminescence using LJL Analyst HT plate reader.

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at -50.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 55.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: 2043-05_Inhibitor_SinglePoint_HTS_Activity

Protocol: 1.#Prepare 1 mM of 2,4-Pyridinedicarboxylic acid (2,4-PDCA) in the Base Buffer (50 mM HEPES pH7.5, 0.01% Tween 20). 2,4-PDCA is used as positive control.

2.#Prepare Enzyme Mix of 20 nM of JMJD2C (BPS Bioscience, 50105), 600 nM of HeK9me3 (Anaspec 64360), 200 uM of ascorbic acid in the Assay Buffer (50 mM HEPES pH7.5, 0.01% Tween 20, 0.01% BSA).

3.#Prepare Cofactor Mix of 100 uM of alpha-ketoglutaric acid potassium salt (2OG), 50 uM Ammonium iron(II) sulfate hexahydrate (Fe(II)) in Assay Buffer.

4.#Onto Aurora 1536-well white high base plates, dispense 100 nL/well of 2,4-PDCA with Combi NL (Thermo) according to plate map.

5.#Dispense 1 uL/well of Cofactor Mix and Enzyme Mix respectively with Combi NL (Thermo) to start the reaction.

6.#Incubate at room temperature for 30 minutes.

7.#Dispense 2 uL/well of Lance Mix (4 nM of Eu-Ab (PerkinElmer), 100 nM of Ulight streptA (PerkinElmer),, 2 mM EDTA, 1x lance buffer (PerkinElmer))

8.#Incubate at room temperature for 60 minutes.

9. Read plate on ViewLux (PerkinElmer) on ex 340/em 618 and em 671.

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Additive)' in Genedata (v10.0.2) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 25.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 0.55.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: 7017-01_Other_SinglePoint_HTS_Activity

Protocol: Protocol:

Cystic Fibrosis airway epithelial cells (CFBE) have been transiently transfected (electroporation) with vectors containing an Yellow Fluoresencent Protein (YFP) halide-sensitive mutant and the human Cystic Fibrosis Transmembrane conductance Regulator with the deletion of the amino acid phenylalanine 508 (CFTR-F508del) provided by Dr. Jinliang Sui from the Flatley Discovery laboratory (Charlestown, MA).

Day 1 (Cell plating and compound pinning in assay plates)

Prior to perform the luciferase reporter assay experiments, the CFBE cells are thawed and reconstituted in MEM medium (Invitrogen, 11965) supplemented with 10% heat inactivated Fetal Bovine Serum (FBS) (Gibco, 16140), 1X antibiotic (Penicillin/Streptamycin/Glutamine) (Gibco, Ref. no. 10378-016). CFBE cells are then plated in 384-well format cell culture treated solid black wall clear bottom assay plates (Corning, Cat. # 3712) at a density of 6,000 cells/well using the multidrop dispenser (standard cassette)(Thermo Scientific) in a final volume of 50 ul. The assay plates are placed in 5% CO2 incubator where they are incubated for 2h at 37oC. After 2h of incubation, the assay plates are pulled out of the incubator and are placed side by side on a pinning table adjacent to compound plates containing the MLPCN library and a sentinel plate containing 32 wells with the positive control C18. 100 nl of the compounds and the positive control C18 (final concentration 10 uM) are then collected on metal pins from these compound plates and transferred to the assay plate. The pins are washed with DMSO and methanol between each transfer. The assay plates are then moved back to the 5% CO2 incubator and incubated for an additional 24h.

Day 2 (Cell wash, CFTR channel activation, quenching and reading on the Flipr)

The assay plates are coming out of the incubator and washed once with PBS (50 ul) using a Biotek plate washer and 20ul of 20 uM Forskolin (Cayman Chemicals) and 3uM potentiator P3 (final concentration) in DPBS is added to the assay plate for 1h at 37oC. Then, the assay plates are cooled down for 30 minutes at room temperature and 25 ul of YFP quencher sodium iodide (72.5 mM final concentration) in DPBS is added and the fluorescence is measured every second for one minute on the Flipr tetra (Molecular Devices).

DPBS (Dulbecco's Phosphate buffered saline)
2.7 mM KCL
8.1 mM Na2PO4
1.5 mM KH2PO4
0.7 mM CaCl2*2H2O
1.1 mM MgCl2

DPBS NaCl
137 mM NaCl

DPBS NaI
145 mM NaI

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 8.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: 7124-02_CD40 Signaling Pathway_CD40 TNFa Counterscreen

Protocol: Protocol:
1) Plate 10 uL RA1-NFkB-luc cells at 12.5K cells/well, using Multidrop Combi reagent dispenser (1250 cells/ul).
2) Add 25 nL compound or positive control, by pin transfer; IKK inhibitor VII will be used at 50 uM.
3) Incubate at 37 masculineC x 1 hour (allowing compounds to enter cells for action).
4) Add TNFa to a final concentration of 10 ng/ml using Multidrop Combi.
5) Incubate at 37 masculineC 4 hrs, then leave plate at RT for 30 minutes.
6) Add 15 uL Steady-Glo luciferase substrate using Multidrop Combi.
7) Incubate at RT for 5min, then read using EnVision plate reader (Perkin Elmer).

Comment: Keywords: CD40 signaling, inhibitors, NF-kB, TNF-alpha

Keywords:
Inhibitors of CD40 Signaling, NF-kB, reporter assay, Luciferase, SteadyGlo, rheumatoid arthritis

PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=32) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.

External ID: 7124-04_CD40 Signaling Pathway_CD40 CellTiter-Glo

Protocol: 1. 10 uL RA1-NFkB-luc cells at 20K cells/well were plated into each well on a 384-well plate using a Multidrop Combi dispenser (Thermo).
2. For each compound, DMSO or positive control IKK inhibitor VII, 50 nL was transferred using a pin tool (Cybio). The final concentration for each compound was 9.4 uM; DMSO 0.5%; and IKK inhibitor VII 50 uM.
3. After cells were incubated at 37C for 1 hour, 10 uL (128 ng/mL) tCD40L was added to make a final concentration of 64 ng/ml.
4. Cells were incubated at 37C for 4.5 hrs before 10 uL 0.5X Cell titer Glo luciferase substrate.
5. Luciferase activity was read after 5 min using EnVision plate reader (Perkin Elmer).

Comment: Keywords: CD40 signaling, inhibitors, NF-kB, tCD40L, viability

Keywords:
Inhibitors of CD40 Signaling, NF-kB, reporter assay, Luciferase, SteadyGlo, rheumatoid arthritis

PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=32) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.

External ID: 7124-01_CD40 Signaling Pathway_Primary Screen 80k Compounds

Protocol: 1) Plate 10 uL RA1-NFkB-luc cells at 20K cells/well, using a Multidrop Combi reagent dispenser (2M cells/mL).
2) Add 2X25 nL compound or positive control, by pin transfer; IKK inhibitor VII will be used at 50 uM.
3) Incubate at 37 masculineC for 1 hour (allowing compounds to enter cells for action).
4) Add 10 uL 128 ng/mL tCD40L per well for a final concentration of 64 ng/ml using a Thermo Multidrop Combi.
5) Incubate at 37 masculineC for 4 hrs, then leave plate at RT for 30 minutes.
6) Add 5 uL SteadyGlo luciferase substrate (Promega) per well using a Thermo Multidrop Combi.
7) Incubate at RT for 5min, then read Luminescence using EnVision (Perkin Elmer) plate reader (Lum 0.2 sec/well).

Comment: Keywords:
Inhibitors of CD40 Signaling, NF-kB, reporter assay, Luciferase, SteadyGlo, rheumatoid arthritis

PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=32) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.

External ID: HTS for inhibitors of DUX4-mediated transcriptional activation

Protocol: Day 1
1. Culture 293T-iDUX4-GR reporter cells with DMEM High Glucose (Hyclone, Cat.No SH30081.01), 10%FBS, 1%glutamax, and 1%Pen/Strep.

2. Plate cells in a 384 well plate (I am currently using Corning 3704 but this plate is discontinued. This plate is replaced with Corning 3570) at 75 cells per ul, 20ul per well using Biomek FX robot and centrifuge 1 min 1000 rpm.

3. Incubate at 37 degrees for 24 hours.


Day 2
4. Treat cells with compounds and then add doxycycline ([C]=50ng/ml) or DMSO for the controls and incubate cells for 24 hours.

Day 3

Plate Reading

Reagents from Targeting Systems:

Kit contains:

A) DLAR-1 1000 (Assay reagent-store at -20 degrees C); 50ml

B) 100x Coelentarazine (Gaussia Substrate- store at -20 degrees C); 500 ul

C) Lysis Buffer (store at -20 degrees C); 30 ml



NOTE: Before reading, make sure reagents are at room temperature before adding, and make sure the plate reader is on and the protocol is set up and ready to read the plate since Gaussia and Red Italica signal get weaker over time (10-15 min after adding reagent is OK).

1. Calculate amount of reagent mix needed for running assay on all wells; 13.3 ul reagent per well for a 384 well plate.

Per well:

DLAR-1: 6.67 uL

Coelentarazine : 0.067 ul

Lysis buffer : 6.67 ul



2. Mix by inverting. This is the reagent mixture.

3. Add 13.3 mul of reagent mixture to each well simultaneously using a 384 well multichannel pipette. Carefully, mix by plate shaker for 5 seconds and spin down 10 sec at 1000 rpm.

4. After 10 min read the plate at 460/40 nm for Gaussia and then read at 620/35 nm for Red italic in Focused Luminescent mode (Z hight: Mid, integration time:100000 mus) using the Analyst AD 96/384 plate reader (LJL Biosystem).

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing 460 nm signal and a smaller or no change in 620 nm signal.

NORMALIZATION:

The raw signals of the plate wells for both channels were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):

The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.

The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.

Experimental wells values were scaled to this range.

All well activities for the 460 nm channel were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention. Resulting negative values were assigned a 0 activity score.

PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:

This was set as equal to the mean of the normalized and corrected sample replicate activities for the 460 nm channel, rounded to the nearest integer .

The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 10.

DIFFERENCE OF NONSPECIFIC VS SPECIFIC CHANNELS:

For each compound the difference between the normalized percent inhibition of the 460 nm and 620 nm channels was determined.

The specificity window for compounds affecting 460 nm but not 620 nm channel was set to a difference of 25 (DIFF)

PUBCHEM_ACTIVITY_OUTCOME:

Samples showing decreasing signal in the 460 nm channel and at least 25 percentage points less activity in the 620 nm channel were assigned an outcome of 2 (active):

PUBCHEM_ACTIVITY_SCORE >= AT, and 460 nm - 620 nm > DIFF

Samples passing showing no activity in the 460 nm channel were assigned an outcome of 1 (inactive):

PUBCHEM_ACTIVITY_SCORE < AT

Samples passing showing activity in both channels were assigned an outcome of 3 (inconclusive) :

PUBCHEM_ACTIVITY_SCORE >= AT, and 460 nm - 620 nm < DIFF

External ID: 2152-01_Activator_SinglePoint_HTS_Activity

Protocol: . Coat plates with 0.1% Gelatin in PBS (20ul per well) for 20 minutes at room temp.
Day 0
. Plate 3,000 HUVEC cells/well in 384 well plates, 50l per well, in EGM-2 medium, incubate at 37 degrees Celsius
Day 2
. Pin 100nl of compound, DMSO
. Add 10 ul Positive Control (TNFalpha at 0.3ng/ml)
. Incubate 16h
Day 3
. Aspirate media using plate washer
. Add 70 ul wash buffer using Thermo Multidrop Combi or plate washer (assay provider uses combi)
. Aspirate wash buffer with plate washer
. Add 20 ul blocking antibody (IgG) with combi
. Incubate at room temp for 15 minutes
. Aspirate
. Add 20 ul Biotin conjugated anti-e-selectin antibody with combi
. Incubate 30 minutes at room temp (or 15 minutes at 37 degrees Celsius)
. Aspirate
. Add 70 ul wash buffer using combi or plate washer (assay provider uses combi)
. Aspirate
. Add 20 ul streptavidin-PE using combi or plate washer (assay provider uses combi)
. Incubate 30 minutes at room temp (or 15 minutes at 37 degrees Celsius)
. Aspirate
. Add 70 ul of PBS (not wash buffer)
. Aspirate
. Add 20ul Hoechst/4% PF/PBS mix (fix cells and stain nuclei)
. Incubate 15 minutes at room temp
. Aspirate (PF is a hazardous waste and is collected in a seperate hazardous waste container)
. Add 70ul PBS
. Aspirate
. Add 70 ul PBS
Plates are sealed with foil seal and stored overnight at 4 degrees Celsius prior to imaging

Plates are imaged on an automated microscope (ImageXpress Micro, Molecular Devices Inc.) at 2x magnification using appropriate filter sets for Hoechst and PE fluorescence.

Cell counts and % of cells positive for PE fluorescence are quantified by using a modified Cell Scoring application module in the image analysis software (MetaXpress, Molecular Devices Inc.)

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
Compounds decreasing cell viability by more than 20 per cent were eliminated. The minimum PUBCHEM_ACTIVITY_SCORE of E-selectin positive cells required for a compound to be called a hit (the activity threshold, or AT) was set at 10.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 60.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: 2142-01_Inhibitor_SinglePoint_HTS_Activity

Protocol: A549 cells were plated at a concentration of approximately 6,000 cells per well in clear-bottom black-walled plates. Compounds were added to the assay plate followed by addition of virus (LREV) that expresses the Venus fluorescent protein early in viral ininfection. During the late viral infection, mCherry was also added to the plate at an MOI of 10 (~ 60,000 plaque forming units (PFU) of virus). Following virus addition, plates were incubated in a tissue-culture incubator for 12-18 hours. At increasing times postinfection, fluorescence from both Venus and mCherrry was determined using a plate-reader and standard conditions for observing Venus fluorescence (EX515/EM530) and mCherry fluorescence (EX587/EM610). In each plate, positive controls (virus infection in the presence of 1% DMSO only) and an inhibitory drug control (AraC at 500 nM) was included and used to normalize the data.
Steps:
1a) A549 cell 4 uL
1b) DMEM medium Control 4 uL
1000 cell/well of A549 ad let cells attach for 3 hrs at 37C in CO2 incubator, DMEM medium in negative control columns
2a) Control Inhibitor
2b) Offline Library 23 nL
23 nL Control Inhibitor, Intraplate 16-pt titration: AraC for mCherry detection & Actinomycin D for Venus detection
3) Viral Infection 2 uL 5 MOI of VAC-LREV virus in DMEM medium is dispensed by Bioraptor
4) Incubation for viral replication Overnight 37C in CO2 incubator
5) Envision Detection Over 18 hrs more Venus read: Exc 510nm/Em590nm & mCherry read: Exc 545nm/Em615nm

Comment: PUBCHEM_ACTIVITY_OUTCOME
Threshold Criteria
<= 33% in the mCherry channel and Venus - mCherry channel must be >= 40%.

Samples passing both criteria are considered Active (2)
Sample passing the 33% threshold for mCherry but not passing the criteria for Venus - mCherry will be considered Inconclusive (3)
Samples passing neither criteria will be considered Inactive (1)
Additionally, samples which pass both criteria but which have unacceptable reproduciblity between replicates will be considered Inconclusive (3)

PUBCHEM_ACTIVITY_SCORE
The score was derived by fitting the mean mCherry values to a 1-100 scale

External ID: 7052-01_Inhibitor_SinglePoint_HTS_Activity_Set2

Protocol: Caspase 6 enzyme was received from collaborator and stored at -80 C. Stock concentration was at 1.2 mg/ml (43.1 uM).

Assay Buffer:
100 mM Hepes pH 7.5, 10% sucrose, 0.1 % CHAPS, 5 mM DTT (final concentration) was added fresh just prior to assay

Reagents:
VEID-R110-VEID: Caspase 6 substrate, was reconstituted to 10 mM from dry powder with DMSO and stored at -20 C.
VEID-CHO: Caspase inhibitor (positive control) was reconstituted to 10 mM from dry powder with DMSO and stored at -20 C.

Assay Ready Plates:
1536 black, solid bottom plates containing 7.5 ul of compound

Protocol:
Add 4 ul 2.2 nM Caspase-6 to 1536 ARPS using BioRaptR
Add 100 nL VEID-CHO to positive control wells using Combi nL
Incubate Room temp 60 min
Add 2 ul of 24 uM VEID-R110-VEID (Caspase 6 substrate) to all wells
Incubate at room temp for 40 min
Read plate on Envision (FITC settings and filters)

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 35.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: 7044-01_Inhibitor_Dose_CherryPick_Activity_Set6

Protocol: 1.) A 10 mM solution of ISGASE(SuK)-AMC is prepared as a DMSO stock solution. This solution is diluted to a 20 uM working solution I with reaction buffer containing 20 mM Tris-HCl, 1 mM dithiothreitol, 100 uM nicotinamide adenine dinucleotide, pH 7.4.
2.) A second working solution is prepared using the 90 uM stock SirT-5 solution by diluting the stock solution using PBS, pH 7.4 to a final concentration of 0.6 uM.
3.) The enzyme reaction is initiated by the simultaneous 2.5 uL additions of working solutions from #1 and #2 above into a 1536-well, black Aurora plate. This results in a reaction with a final substrate ISGASE(SuK)-AMC concentration of 10 uM and a final SirT-5 concentration of 0.3 uM.
4.) The reaction is allowed to proceed for 1 hr. at room temperature.
5.) 10X Trypsin (0.5% Trypsin-EDTA) is diluted to 1X (0.05%) by the addition of H20. 2.5 uL of 1X trypsin is then added to the reaction and incubated at room temperature for 1 hr (final trypsin concentration 0.0167% = 0.33X).
6.) After the 1 hr. incubation, the reaction progress is evaluated by monitoring the fluorescence emission at 460 nm. This can be done using an Envision with 50/50 splitter, Ex. 380 nm and Em 460 nm. Or using the View Lux equipped with the same filter set.

* Human SirT 5 was expressed and purified by the Lin laboratory at Cornell.
* ISGASE(SuK) was synthesized and purified by the Lin laboratory at Cornell.
* ISGASE(K) was synthesized and purified by the Lin laboratory at Cornell
* Suramin*hexasodium was purchased from Enzo, cat. # ALX-430-022
* 0.5% Trypsin-EDTA (10X) was purchased from Gibco (Life Technologies), cat. # 15400-054

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC; n=128) and positive control wells (PC; n=128) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.